Hi,
I'm having problems with my pET system. I've successfully managed to
ligate an insert coding for the 24kDa protein I want to express into
pet15b. I then transformed the plasmid into BL21(DE3)pLysS, added 1mM
IPTG, and waited for over-expression. Everything seemed to be working -
nice over-expression of a protein of about the right size which purified
out fairly easily on a His.Bind column (though it did elute at fairly low
levels of imidazole). I thought all this was too easy to be true, and it
was. When we sequenced the peptide it turned out to be chloroamphenicol
acetyl transferase - a protein which is expressed by the pLysS plasmid.
No sign of my protein at all, and inducible over-expression of an
antibiotic-resistance protein (no sign of CAT if I don't add IPTG). Not
what I wanted!
Has anybody had any similar experiences or any ideas what may be going
on? Any suggestions would be very gratefully received,
Thanks,
Jared
--
Jared Head at the Department of Biochemistry, University of Bristol
jared.head at bristol.ac.uk